JUN mRNA translation regulation is mediated by multiple 5’ UTR and start codon features

Regulation of mRNA translation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5′ untranslated region (5′ UTR) and with the 5′ -7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5′ UTR is nearly one kilobase in length, and has a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Here we find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5′ UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Our results reveal additional layers of regulation for JUN translation and show the potential of JUN as a model transcript for understanding multiple interacting modes of translation regulation.

1.In Fig 1, authors identified several important sequences by mutagenesis, which regulate JUN mRNA translation which are interesting, especially, mutants A, F, and G. i) It will be interesting to know what happens to a double mutant of F with A or G. Do any of mutation A or G overcome the mutation F? ii) In mutant F, do eIF3 still binds to stem loop?iii) Do mutants A and G disrupt the binding of eIF3?
We thank the reviewer for raising these interesting questions.We agree it would be interesting to test the effects of these double mutants, and how they affect eIF3 binding.We think this would be a valuable question to explore in future studies, and now mention this in the Discussion.

In the presence of any of the mutations mentioned in Fig 1, does overexpression of eIF4A overcome the translation repression?
This is an interesting question.As noted in the Discussion, RocA inhibits translation of the JUN reporter mRNA, even with the SL mutation.This suggests that the eIF4A dependence of JUN translation is at least partially independent of the eIF3 binding requirement.We think this question would be interesting to explore in a future study.We think this would be an interesting question to test in a future study.As noted above, inhibition of JUN translation by RocA seems to be independent of the action of eIF3, at least with respect to the SL region.We've modified the Discussion to address this point more clearly.5. Lee et al. (2016) show that the JUN mRNA translation initiation complex lacks eIF4F components.Here, authors showed that eIF4A plays a role in JUN mRNA translation.Can please explain this discrepancy?
Yes, this is a surprising result of our studies.While our experiments evaluating the role of eIF4A in JUN mRNA translation were done mainly in cells, the experiments presented by Lee et al (2016) were performed using in vitro translation reactions and sucrose gradients.In the sucrose gradient experiment, Dr. Lee isolated 48S-like complexes for her analysis.We think this represents a very late stage of translation initiation, and eIF4A may be acting "upstream" of this event.This will be a fascinating question to explore in the future.We've made sure the Discussion captures this idea.
6.In Fig 2C and 2D, results are not matching for DMSO treatment.Why does the ∆SL mutant not exhibit a reduction in the 'in-vitro translation' sample, whereas the CAAC+∆SL mutant does show a reduction?
Recapitulating the translational repression for the JUN 5' UTR ∆SL reporter construct has been challenging in vitro.It is possible that the mechanism that regulates translation of a reporter mRNA containing this 5' UTR requires elements predominantly active in vivo.We've raised this point in the Discussion.
7. Why are two start codons of JUN mRNA significant, as they are only 4 codons apart.
Thanks for raising this question.Previous studies have shown a role for upstream start codons (uAUGs) that are located in close proximity to the downstream-or main-start codon (mAUG) (Smith et al. 2005;Matsuda and Dreher 2006).In this scenario, leaky scanning of uAUGs in a weak translational context allows for backward oscillation of the ribosome and increases translation of the mAUG.This constitutes an alternative mode of translation for mRNAs with these types of start codons.We hypothesize that this could be the case for the JUN mRNA, especially given that both start codons and their translational contexts are conserved.We describe these ideas in the Discussion.

Reviewer 2
This manuscript addresses processes that can contribute to c-Jun mRNA translation.Prior reports has documented that Jun mRNA translation is mediated by its direct engagement with eIF3.This manuscript aims to provide some more details to this translational control process, notably some features of the secondary structure of Jun mRNA, and its potential sensitivity to an anti-cancer drug RocA, which targets eIF4A.These goals are met in this study and the manuscript is clearly written and experimental conclusions are supported by rigorous experiments.There are a few concerns, none major, that should be addressed to enhance rigor and clarity.
3. InFig 2A, "JUN 5' UTR G" wrongly labelled, it is "JUN 5' UTR E".Thank you for pointing this out!We've corrected this in the figure.4. In Fig 2A, since mutant A and G are unreactive by SHAPE, also showed significant increase in JUN mRNA translation, do any of these mutations show resistance to RocA treatment?